Detecting targets using nucleic acids having both a variable region and a conserved region

ABSTRACT

The invention relates to nucleic acid molecules for use in detecting a target nucleic acid molecule which is a member of a class of nucleic acid molecules and which is characterised by a specific variant region, said nucleic acid molecule comprising (i) a nucleic acid stem region which comprises a nucleic acid interaction site directed to a conserved region of the class of which said target nucleic acid molecule is member, or part thereof and which conserved region is located proximally to a variant region; operate y linked to (ii) a nucleic acid recognition region comprising at least two nucleotides. The nucleic acids are used in arrays and are an efficient means of screening molecules exhibiting a unique nucleotide sequence within a randomly varying population. The invention is useful in monitoring the effectiveness of therapeutic drug therapies and the progression of medical conditions, characterised by the presence of clonal populations of cells, particularly clonal lymphocyte populations.

FIELD OF THE INVENTION

The present invention is directed to a novel array of oligonucleotidesand methods for use thereof. More particularly, the present invention isdirected to a novel array of amplification primers which facilitate theamplification of a specific nucleotide molecule of interest from apopulation of molecules, which vary randomly in sequence from onemolecule to the other, in an efficient manner. The design of this arrayhas now facilitated the development and implementation of very efficientmeans of screening for molecules exhibiting a unique nucleotidesequences within a randomly varying population. Accordingly, themolecules of the present invention are useful in a range of applicationsincluding, but not limited to, monitoring the progression of a conditioncharacterised by the presence of a clonal population of cells, inparticular a clonal lymphocyte population, monitoring the levels of aclonal cell population, predicting the likelihood of a subject's relapsefrom a remissive state to a disease state or for assessing theeffectiveness of existing therapeutic drugs and/or new therapeuticdrugs.

BACKGROUND OF THE INVENTION

The reference to any prior art in this specification is not, and shouldnot be taken as, an acknowledgment or any form of suggestion that thatprior art forms part of the common general knowledge in Australia.

Lymphocytes, the cells that subserve the immune response, are of twotypes—B lymphocytes which produce antibody and T lymphocytes which areinvolved in cellular immunity. During development, in order to develop aspecific immune response, each lymphocyte rearranges in a unique fashionone or a few specific genes—the immunoglobulin genes for a B lymphocyteand the T cell receptor genes for a T lymphocyte. All descendants ofthese lymphocytes will then carry the same rearrangement.

A neoplasm is believed to arise as the result of summation of geneticchanges in a single cell. Subsequent proliferation of that cell givesrise to a population of descendants. Neoplasms arising from malignantchange in a B or T lymphocyte (often referred to as “cancers”) wouldtherefore predominantly comprise a clone of cells, each of whichcontains the same gene rearrangements that were present in the foundercell, although secondary gene rearrangements may occur within theneoplastic clone leading to genetically different subclones.

Lymphocytic neoplasms are therefore clonal disorders. They develop afterone or more mutations in a single cell cause the cell and its progeny tomultiply progressively and exponentially. For example, when lymphocyticleukemic clones number 10¹¹ to 10¹² cells in the body, clinical symptomsensue. Without treatment, the clone continues to expand, and deathresults when there are approximately 10¹³ leukemic cells. If, however,the patient receives cytotoxic treatment, the clone decreases in size,and it can no longer be identified by conventional techniques when itcomprises fewer than about 10¹⁰ cells. At this point, the patient isjudged to be in clinical and haematological remission, although the term“remission”, in fact, refers only to a somewhat arbitrary point towardone end of a continuum of leukemic-cell number. Since the number ofleukemic cells that may remain during remission is unknown and may rangefrom 0 to 10¹⁰, treatment after remission has been achieved is empiricaland its intensity is based on various clinical or laboratory prognosticfactors determined at diagnosis or early in treatment. Consequently,some patients may receive too little treatment and others may receivetoo much.

Current methods for monitoring malignant lymphocytes involve the use ofa “marker” which is shared by all cells of the clone. The marker may bea surface antigen, or patterns of several surface antigens, or it may bea molecular change. The molecular changes which are used may be broadlyclassified into two types—those which involve a chromosomaltranslocation or inversion, and those which will use the immunoglobulinor T cell receptor (herein referred to as “TCR”) gene arrangements.

In the context of screening for immunoglobulin or TCR generearrangements, as a means of analysing a particular clonal cellpopulation, variability exists in the nucleotide sequences of therearranged variable region of a population of B cells or T cells. Onoccasions the variability may involve one or only a few bases, when forexample polymorphisms exist in the population. Under such circumstancesit may be feasible to amplify from these slightly variable regions byhaving a small panel, usually one or several, PCR primers, which areusually constructed to bind to the range of known variants. However, inmany situations the variability is too great and, in amplifying aparticular sample, the conventional approach is therefore to sequencethe region of interest and synthesise a specific primer or primers whichbinds to the particular sequence of the region of interest. Until theadvent of the present invention, however, it has not proved practical topre-synthesise primer arrays for ongoing use in amplifying nucleic acidmolecules of interest from a class of molecules exhibiting regions ofextensive variability, owing to the number of primers which would berequired. For example, in considering a region of n nucleotides, each ofwhich may be adenine, guanine, cytosine or thymine, the number ofpossible combinations is 4^(n).

Although the technology for synthesising a specific primer is widelyavailable, the time and cost involved in doing so can become prohibitivewhere one is required to use many different primer sequences, forexample, where a clinical or diagnostic laboratory requires access to aunique primer for each individual patient who is under investigation. Asdetailed above, to pre-synthesise an array of primers, for repeated useas a primer source, from which a suitable primer could be selected forthe amplification of a region of high variability, the large number ofprimers which would be required to be synthesised is prohibitive both interms of cost and practicality. Accordingly, there is a need to developmeans of facilitating the efficient and routine amplification of aspecific marker sequence for any given patient.

In work leading up to the present invention, the inventors havedeveloped a means of designing a panel of pre-synthesisedoligonucleotide primers from which one can select an appropriateoligonucleotide primer for amplifying a specific sequence of DNA, suchas a neoplastic lymphoid cell rearranged immunoglobulin or TCR generegion, which randomly varies from one cell population to the next.

The invention has been developed in light of the determination that atarget nucleic acid molecule comprising the feature that a variantnucleotide region (as between members of the class to which it belongs)is positioned adjacent to a substantially invariant (conserved)nucleotide region can be selected for by primers which are specificallydesigned to exploit this feature. The nature of the design of this arrayof oligonucleotides can facilitate the amplification of a targetnucleotide sequence, even where a very high degree of nucleotidevariation exists across different members of the class to which itbelongs, without the need to synthesise a prohibitively large panel ofoligonucleotides for ongoing use. In one example, the primers comprisingan array are designed with a 5′ stem region, which recognises thesubstantially conserved region of the target nucleic acid moleculeabutting the variant region, and a 3′ recognition end which comprises arandom combination of two or more nucleotides, and which will interactwith a complementary variant region. Preferably, the array comprisesprimers which correspond to each of the possible 16, 64 or 256combinations of nucleotides for the 3′ recognition end, where that 3′end consists of 2, 3 or 4 nucleotides, respectively for example, if the3′ recognition end comprises more than 4 nucleotides, then the number ofprimers constituting the array will increase. By selecting and utilisingan appropriately designed oligonucleotide from the array, either inisolation or in tandem with another suitable oligonucleotide, ashereinafter explained in more detail, any target nucleic acid sequencevariant of interest can ultimately be selectively amplified. Thedevelopment of these oligonucleotide arrays now facilitates thedevelopment of cost effective, rapid and routine means of screening forany one or more specific target nucleic acid molecules within a class ofnucleic acid molecules which are defined by a substantially conservednucleotide sequence region positioned adjacent to a variable sequenceregion.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integers or steps.

The subject specification contains nucleotide sequence informationprepared using the programme PatentIn Version 3.1, presented hereinafter the bibliography. Each nucleotide sequence is identified in thesequence listing by the numeric indicator <210> followed by the sequenceidentifier (eg. <210>1, <210>2, etc). The length, type of sequence (DNA,etc) and source organism for each nucleotide sequence is indicated byinformation provided in the numeric indicator fields <211>, <212> and<213>, respectively. Nucleotide sequences referred to in thespecification are identified by the indicator SEQ ID NO: followed by thesequence identifier (eg. SEQ ID NO:1, SEQ ID NO:2, etc.). The sequenceidentifier referred to in the specification correlates to theinformation provided in numeric indicator field <400> in the sequencelisting, which is followed by the sequence identifier (eg. <400>1,<400>2, etc). That is SEQ ID NO: 1 as detailed in the specificationcorrelates to the sequence indicated as <400>1 in the sequence listing.

One aspect of the present invention is directed to an array of isolatednucleic acid molecules or derivatives or analogues thereof, for use indetecting a target nucleic acid molecule which is a member of a class ofnucleic acid molecules and which is characterised by a specific variantregion said nucleic acid molecules comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof and which substantially conserved region is    located proximally to a variant region operably linked to-   ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said nucleic acid molecules comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said nucleic acid molecules optionally comprise one or more    universally hybridising bases, or analogues thereof, intervening    said stem region and said recognition region.

Another aspect of the present invention is directed to an array ofisolated oligonucleotides or derivatives or analogues thereof, for usein detecting a target nucleic acid molecule which is a member of a classof nucleic acid molecules and which is characterised by a specificvariant region, said oligonucleotides comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said oligonucleotides comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said oligonucleotides optionally comprise one or more universally    hybridising bases, or analogues thereof, intervening said stem    region and said recognition region.

Yet another aspect of the present invention is directed to an array ofisolated oligonucleotide primers or derivatives or analogues thereof foruse in detecting a target nucleic acid molecule which is a member of aclass of nucleic acid molecules and which is characterised by a specificvariant region, said oligonucleotide primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said oligonucleotide primers comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said oligonucleotides optionally comprise one or more universally    hybridising bases, or analogues thereof, intervening said stem    region and said recognition region.

Still another aspect of the present invention provides an array ofisolated DNA primers or derivatives or analogues thereof, for use indetecting a target gene which is a member of a class of genes which arecharacterised by a specific variant region said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target gene, or part thereof, is a    member, which substantially conserved region is located proximally    to said variant region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Yet still another aspect of the present invention is directed to anarray of isolated DNA primers or derivatives or analogues thereof foruse in detecting a rearranged TCR or immunoglobulin variable genesegment, or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the TCR or immunoglobulin variable gene segment and which    substantially conserved region is located proximally to a variant    region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Still yet another aspect of the present invention is directed to anarray of isolated DNA primers or derivatives or analogues thereof foruse in detecting a rearranged TCR or immunoglobulin variable genesegment, or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    portion of the 5′ end of the antisense strand of the V gene segment,    or part thereof, operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

A further aspect of the present invention is directed to an array ofisolated DNA primers or derivatives or analogues thereof for use indetecting a rearranged TCR or immunoglobulin variable gene segment, orpart thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    portion of the 5′ end of the antisense strand of the D gene segment,    or part thereof, operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

In yet another preferred embodiment of the present invention is directedto an array of isolated DNA primers or derivatives or analogues thereoffor use in detecting a rearranged TCR or immunoglobulin variable genesegment, or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the 5′ end of the sense strand of the J gene segment, or    part thereof; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Another further aspect of the present invention provides an array ofisolated DNA primers or derivatives or analogues thereof, for use indetecting a rearranged TCR or immunoglobulin variable gene segment, orpart thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to the 5′ end of the    antisense strand of the V gene segment, or part thereof; operably    linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more inosines, or analogues thereof,    intervening said stem region and said recognition region.

In another aspect there is provided an array of isolated DNA primers orderivatives or analogues thereof, for use in detecting a rearranged TCRor immunoglobulin variable gene segment, or part thereof, said primerscomprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to the 5′ end of the    antisense strand of the D gene segment, or part thereof; operably    linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides wherein said primers comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said primers optionally comprise one or more inosines, or analogues    thereof, intervening said stem region and said recognition region.

In still another preferred embodiment there is provided an array ofisolated DNA primers or derivatives or analogues thereof, for use indetecting a rearranged TCR or immunoglobulin variable gene segment, orpart thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to the 5′ end of the sense    strand of the J gene segment, or part thereof; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more inosines, or analogues thereof,    intervening said stem region and said recognition region.

In yet another further aspect the present invention provides an array ofisolated DNA primers or derivatives or analogues thereof, for use indetecting a target nucleic acid molecule which is a member of a class ofnucleic acid molecules which are characterised by a specific variantregion sequence, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising three nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise at least two inosines, or analogues thereof,    intervening said stem region and said recognition region.

In still another further aspect there is provided an array of isolatedDNA primers or derivatives or analogues thereof, for use in detecting atarget nucleic acid molecule which is a member of a class of nucleicacid molecules which are characterised by a specific variant regionsequence, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    regions of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising four nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise at least two inosines, or analogues thereof,    intervening said stem region and said recognition region.

Another aspect of the present invention is directed to a method ofidentifying a target nucleic acid molecule in a sample, which moleculeis a member of a class of nucleic acid molecules characterised by aspecific variant region sequence, said method comprising

-   (i) contacting said sample with an oligonucleotide as hereinbefore    defined for a time and under conditions sufficient to facilitate    interaction of said oligonucleotide with said target nucleic acid    molecule;-   (ii) amplifying said nucleic acid target; and-   (iii) optionally consecutively repeating said amplification steps    utilising the nucleic acid material amplified in the preceding step    together with a leap frog oligonucleotide; and-   (iv) detecting said amplified product.

Another aspect of the present invention provides a method of detectingand/or monitoring a clonal population of cells in a mammal, which clonalcells are characterised by a target nucleic acid molecule which is amember of a class of nucleic acid molecules characterised by a specificvariant region sequence, said method comprising:

-   (i) contacting the nucleic acid material of a biological sample    derived from a mammal with an oligonucleotide as hereinbefore    defined for a time and under conditions sufficient to facilitate    interaction of said oligonucleotide with said target nucleic acid    molecule;-   (ii) amplifying said nucleic acid target;-   (iii) optionally consecutively repeating said amplification steps    utilising the nucleic acid material amplified in the preceding step    together with a leap frog oligonucleotide; and-   (iv) detecting said amplified product.

Accordingly, still another aspect of the present invention is directedto a method for diagnosis of the onset of or a predisposition to theonset of a disease condition or for monitoring or prognosing theprogression of a disease condition in a mammal, which condition ischaracterised by the presence or change in the level of a target nucleicacid molecule, or clonal cell population characterised by a targetnucleic acid molecule, which molecule is a member of a class nucleicacid molecule characterised by a specific variant region sequence, saidmethod comprising:

-   (i) contacting a sample derived from said mammal with an    oligonucleotide as hereinbefore defined, for a time and under    conditions sufficient to facilitate interaction of said    oligonucleotide with said target nucleic acid molecule;-   (ii) amplifying said nucleic acid target;-   (iii) optionally consecutively repeating said amplification steps    utilising the nucleic acid material amplified in the preceding step    together with a leap frog oligonucleotide; and-   (iv) detecting said amplified product.

Yet another aspect of the present invention is directed to a kit forfacilitating the identification of a target nucleic acid molecule, saidkit comprising compartments adapted to contain any one or more of theoligonucleotide primers as hereinbefore defined, reagents useful forfacilitating interaction of said primer with the target nucleic acidmolecule and reagents useful for enabling said interaction to result inamplification of said nucleic acid target. Further compartments may alsobe included, for example, to receive biological or non-biologicalsamples.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the effect of the number ofinosines and the temperature of annealing on the degree of amplificationachieved by 20 cycles of PCR.

FIG. 2 is a graphical representation of the observed vs expected minimalresidual disease (“MRDI”).

FIG. 3 is a schematic representation of an example of a primary primerand leap frog primer used to amplify the desired nucleotide sequence ina specific fashion. The primary primer and the leap frog primer are eachchosen from arrays, which in this case, each comprise 64 (4⁴) members.The stem region (solid line) and the intervening region (dashed line),which shows some variability, are shown, together with the next 8 basesof all possible sequences. The bases of the sequence which it isdesigned to amplify are shown in bold and the sequence recognised isACGTTCAG. The nucleic acid molecules which it is desired to detect arethose which contain this sequence.

FIG. 4 is a graphical representation of the paired comparisons ofperformance of 10 inosine primers with 10 standard primers. For eachpair the standard primer had exactly the same sequence as the inosineprimer except that the 6 inosines were replaced by the 6 bases actuallypresent in the particular gene rearrangement. Rearrangements from 10leukaemic marrow samples at diagnosis (closed symbols) and from 10peripheral blood samples from normal individuals (open symbols) werestudied by quantitative PCR with the end-point of cycles to threshold(Ct). The inosine primers showed essentially the same Ct values as thestandard primers, indicating that they amplified with the sameefficiency. The Cts for the monoclonal leukaemic samples were 6-12cycles less than for the polyclonal peripheral blood samples. Thisdifference indicates good specificity and is of the order of differenceexpected, as all of the rearrangements in leukemic DNA would be expectedto amplify whereas approximately only 1 in 4⁴ ie, 1 in 256, of therearrangements in peripheral blood DNA would be expected to do so.

FIG. 5 is a graphical representation of the results of measurement ofminimal residual disease (MRD, the proportion of leukaemic cells) in themarrow in 8 adults with acute lymphoblastic leukaemia. The patients wereparticipating in a trial carried out by the Australasian Leukaemialymphoma Group which was investigating the utility of a new form of drugtreatment. Bone marrows were done at diagnosis and on days 28 and 56 oftreatment. MRD estimations on days 28 and 56 were performed using 3rounds of PCR, the last round being quantitative real-time PCR. Thefirst round used V and J primers specific for the IgH rearrangementinvolved, the second round used primers containing 6 inosines andterminating at the 3′ end with the 4 patient-specific nucleotides, andthe third round used either a D-region specific primer, a specificleap-frog inosine-containing primer, or a patient-specific primer. Whenan inosine primer was used, annealing was performed at 43 deg.C.Experiments have shown that good specificity and sensitivity is obtainedat this temperature.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination thatone can design an array of pre-synthesised oligonucleotide primers fromwhich can be selected a primer capable of amplifying any one of themembers of a class of nucleic acid molecules which exhibit a region ofnucleotide sequence variation adjacent to a region of substantialconservation of sequence (such as occurs in the context of rearrangedTCR or immunoglobulin genes). Importantly, this can be efficientlyeffected without the need to necessarily generate the prohibitivelylarge numbers of distinct primers which, to date, would have beenrequired to provide an effective means of achieving this type of targetnucleic acid selection. Specifically, due to the design of primers whichexploit the region of the target sequence where the substantiallyinvariant/conserved sequence abuts the variant sequence, a suitableprimer can be selected from an array of as few as 4²-4⁴ oligonucleotidesto achieve highly specific selection either via a single amplificationstep or by consecutive amplification steps which use primersappropriately selected from two or more distinct arrays. This system ofconsecutive amplification facilitates a high level of sensitivity and ishereinafter described in more detail. The development of these arrayshas now facilitated their application to detecting or monitoringconditions characterised by the presence of cells, in particular clonalpopulations of cells, expressing a nucleic acid molecule exhibiting thistype of structure.

Accordingly, one aspect of the present invention is directed to an arrayof isolated nucleic acid molecules or derivatives or analogues thereof,for use in detecting a target nucleic acid molecule which is a member ofa class of nucleic acid molecules and which is characterised by aspecific variant region said nucleic acid molecules comprising:

-   i) a nucleic acid stem region, which stem region comprises a nucleic    acid interaction site directed to a substantially conserved region    of the class of which said target nucleic acid molecule is a member,    or part thereof and which substantially conserved region is located    proximally to a variant region operably linked to-   ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said nucleic acid molecules comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said nucleic acid molecules optionally comprise one or more    universally hybridising bases, or analogues thereof, intervening    said stem region and said recognition region.

Reference to a “nucleic acid molecule” for use in “detecting a target”should be understood as a reference to a nucleic acid molecule orderivative or analogue thereof which functions to identify, isolateand/or enrich a target molecule. Examples of such molecules include, butare not limited to, nucleic acid molecules which can function as probesand/or amplification primers. Preferably, the subject nucleic acidmolecule is an oligonucleotide of 4 to 60 nucleotides in length,preferably 10 to 50 in length, more preferably 15 to 45 in length, stillmore preferably 20 to 40 in length, yet more preferably 25 to 35 inlength. Most preferably, said nucleic acid molecule is about 26, 27, 28,29, 30, 31, 32, 33 or 34 nucleotides in length.

More particularly, the present invention is directed to an array ofisolated oligonucleotides or derivatives or analogues thereof, for usein detecting a target nucleic acid molecule which is a member of a classof nucleic acid molecules and which is characterised by a specificvariant region, said oligonucleotides comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said oligonucleotides comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said oligonucleotides optionally comprise one or more universally    hybridising bases, or analogues thereof, intervening said stem    region and said recognition region.

Preferably, said oligonucleotide is an oligonucleotide primer.

Accordingly, still more particularly, the present invention is directedto an array of isolated oligonucleotide primers or derivatives oranalogues thereof for use in detecting a target nucleic acid moleculewhich is a member of a class of nucleic acid molecules and which ischaracterised by a specific variant region, said oligonucleotide primerscomprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said oligonucleotide primers comprise unique nucleic acid    recognition region sequences relative to one another and wherein    said oligonucleotides optionally comprise one or more universally    hybridising bases, or analogues thereof, intervening said stem    region and said recognition region.

Reference to a “nucleic acid” or “nucleotide” should be understood as areference to both deoxyribonucleic acid or nucleotides and ribonucleicacid or nucleotides or derivatives or analogues thereof. In this regard,it should be understood to encompass phosphate esters of ribonucleotidesand/or deoxyribonucleotides, including DNA (cDNA or genomic DNA), RNA,mRNA or tRNA among others. The nucleic acid molecules of the presentinvention may be of any origin including naturally occurring (such aswould be derived from a biological sample), recombinantly produced orsynthetically produced.

Reference to “derivatives” should be understood to include reference tofragments, parts, portions, homologs and mimetics of said nucleic acidmolecules from natural, synthetic or recombinant sources. “Functionalderivatives” should be understood as derivatives which exhibit any oneor more of the functional activities of nucleotides or nucleic acidmolecules. The derivatives of said nucleotides or nucleic acid sequencesinclude fragments having particular regions of the nucleotide or nucleicacid molecule fused to other proteinaceous or non-proteinaceousmolecules. “Analogs” contemplated herein include, but are not limitedto, modifications to the nucleotide or nucleic acid molecule such asmodifications to its chemical makeup or overall conformation. Thisincludes, for example, modification to the manner in which nucleotidesor nucleic acid molecules interact with other nucleotides or nucleicacid molecules such as at the level of backbone formation orcomplementary base pair hybridisation. The biotinylation of a nucleotideor nucleic acid molecules is an example of a “functional derivative” asherein defined. Derivatives of nucleic acid molecules may be derivedfrom single or multiple nucleotide substitutions, deletions and/oradditions. The term “functional derivatives” should also be understoodto encompass nucleotides or nucleic acid exhibiting any one or more ofthe functional activities of a nucleotide or nucleic acid sequence, suchas for example, products obtained following natural product screening.

Reference to an “oligonucleotide primer” or “primer” should beunderstood as a reference to any molecule comprising a sequence ofnucleotides, or functional derivatives or analogues thereof, thefunction of which includes the hybridisation of at least one region ofsaid nucleotide sequence with a target nucleic acid molecule and theamplification of said target sequence. Accordingly, reference to a“target nucleic acid molecule” is a reference to any molecule comprisinga sequence of nucleotides or functional derivative or analogue thereofwhich molecule is a molecule of interest and is therefore the subject ofidentification via an amplification step. Preferably, the target nucleicacid molecule is a gene, or part thereof, such as one or more of thejunction regions of the rearranged V, D or J segments of the TCR orimmunoglobulin genes.

Both the primer and the target nucleic acid molecule may comprisenon-nucleic acid components. For example, the primer may also comprise anon-nucleic acid detection tag (for example, allowing it to additionallyor alternatively function as a probe), such as a fluorescent tag or someother non-nucleic acid component which facilitates the functioning ofthe molecule, such as the detection or immobilisation of the molecule.Similarly, the target nucleic acid molecule may comprise a non-nucleicacid component. For example, the target nucleic acid molecule may bebound to an antibody. This may occur, for example, where the targetnucleic acid molecule is present in a biological sample isolated from anindividual who is mounting an immune response, such as an autoimmuneresponse, to said target nucleic acid molecule. In another example, theprimer may be a protein nucleic acid which comprises a peptide backboneexhibiting nucleic acid side chains. Preferably, said target nucleicacid molecules is a gene region and said oligonucleotide primer is a DNAprimer.

The present invention therefore preferably provides an array of isolatedDNA primers or derivatives or analogues thereof, for use in detecting atarget gene which is a member of a class of genes which arecharacterised by a specific variant region said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target gene, or part thereof, is a    member, which substantially conserved region is located proximally    to said variant region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

It should be understood that the phrase “characterised by” is intendedto indicate that the subject target nucleic acid molecule exhibits thedefined characteristic but it is not intended as a limitation in respectof what other characteristics the molecule might also exhibit. It shouldalso be understood that the subject characteristic is not necessarilyuniquely exhibited only by the subject target molecule although in apreferred embodiment the characteristic is one which identifies themolecule of interest from the molecules of non-interest which arepresent in a sample.

The present invention is predicated on the finding that some classes oftarget nucleic acid molecules (for example, certain classes of genes)are characterised by a nucleotide sequence which comprises asubstantially conserved region of sequence which is located proximallyto a region which exhibits some degree of variation of sequence from onemolecule to another. This may occur, for example, in genes which exhibitpolymorphic variations. In another example, genes which undergorearrangements, such as the TCR and immunoglobulin genes fall into thisclass. In yet another example, the target variation may be the result ofnon-natural gene mutation events such as recombinant engineering of agene or random mutation due to toxic environmental factors. In thisregard, reference to a “class” of molecules should be understood as areference to a group of nucleic acid molecules, preferably genes, whichexhibit a level of sequence homology high enough that they can becharacterised as members of a single class of molecules (such as asingle class of gene) but which members nevertheless exhibit uniquedifferences in regions of their actual nucleic acid sequences.Preferably, the subject sequences are of the class of rearranged or atleast partly rearranged immunoglobulin or TCR variable receptor genefamilies.

Accordingly, in a preferred embodiment, the present invention isdirected to an array of isolated DNA primers or derivatives or analoguesthereof for use in detecting a rearranged TCR or immunoglobulin variableregion gene, or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the TCR or immunoglobulin variable region gene and which    substantially conserved region is located proximally to a variant    region; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Reference to a “substantially conserved region” should be understood asa reference to a region located proximally to the variant region andwhich is characterised by sufficient consensus sequence between themembers of the class in issue that a primer can be designed which willbind most, preferably all, the members of the subject class at theconserved region. The subject conserved region is located proximally toa region which exhibits some degree of sequence variation. Withoutlimiting the present invention to any one theory or mode of action, itis the existence and detection of the region of variation which enablesthe identification of a unique molecule within the class of nucleic acidmolecules. Reference to “variant region” should therefore be understoodas a reference to a region of nucleotides which exhibit variation ofsequence between members of the class to which they belong. The degreeof variation which exists in the context of a given class of moleculesmay in itself show significant differences between different classes ofmolecules. For example, the variation may involve one or only a fewnucleotides, such as occurs in the context of a gene or class of geneswhich are subject to polymorphic variations. In other situations,however, the variation may be great. Accordingly, it should beunderstood that the “classes” of molecules to which this invention isdirected may take any one of a number of forms including that the classcorresponds to a family of genes one or more of which are present in thegenome of the cells of a single individual, a cluster of genes which arethe result of gene rearrangement, one or more of the rearranged genesbeing present in the genomes of specific cell populations of anindividual or a single gene which exists in various polymorphic formsone or more of which forms can be found either within the genome of oneindividual (such as the MHC genes) or between individuals. It shouldalso be understood that the variation may occur either as the result ofnaturally occurring molecular events, such as gene rearrangement orsomatic mutations or it may be artificially induced such as can occurwith exposure to chemicals, radiation or molecular engineering, forexample.

It should also be understood that the subject conserved region andvariant region may be arranged in any orientation. That is, the variantregion may be located 3′ to the conserved region on the sense strand ofthe gene of interest or the variant region may be located 5′ to theconserved region of the sense strand. When one considers the antisensestrand of the gene of interest, these 5′/3′ directional positions arereversed. For example, in the context of the sense strand of theimmunoglobulin gene, the conserved V gene segment and the 3′ end of theD gene segment are positioned 5′ to the region of variation which abutsboth these segments. However, the J segment and the 5′ end of the Dsegment of the sense strand are positioned in the 3′ direction relativeto the region of variation. With respect to the antisense strand, these5′ and 3′ directional positions are reversed, as detailed below:

Accordingly, the particular strand of the double stranded target nucleicacid molecule to which the oligonucleotide primer of the presentinvention is directed will depend on the 5′/3′ orientation of theconserved and variant regions of the target nucleic acid molecule. Ingeneral, where one is seeking to detect a target molecule in which thevariant region is located in the 3′ direction to the conserved region,such as occurs with the V segment of the immunoglobulin gene, it will benecessary to design a panel of primers which are directed to theantisense strand of this gene segment since this will facilitateextension of the primer from its 3′ end, that is from the terminal endof the recognition region of the primer. Accordingly, it should beunderstood that where the variant region of a target gene is located 5′to the conserved region, such as occurs with the J segment of theimmunoglobulin gene, the primer is designed such that it is directed tothe sense strand of a target nucleic acid molecule.

Reference to the nucleic acid molecule which is detected using theprimers of the present invention being “characterised by” a specificvariant region should be understood as a reference to the sequence ofthe variant region being found in that nucleic acid molecule but whichvariant region is either not found in other nucleic acid molecules ofthat class or is not found at a significant level in other nucleic acidmolecules. By “significant” is meant that the amplification of apopulation of nucleic acid molecules utilising a primer directed to thatvariant region nevertheless provides a useful indicator of the nucleicacid molecule of interest based either on a single step amplificationreaction or consecutive reactions, as hereinafter described in moredetail.

The primers of the present invention are directed to identifying anucleic acid molecule which is characterised by a substantiallyconserved region located proximally to a variant region. By “proximallyto” is meant that the regions are positioned relative to one anothersuch that a primer can be designed to interact with both regions.Preferably, the variant region is immediately adjacent to the terminalend of the conserved region. However, it may also be located near to theconserved region and therefore separated by a number of interveningnucleotides.

In a preferred embodiment of the present invention, the nucleic acidmolecule to which an array of primers is generated is the genomicrearranged variable region of the TCR or immunoglobulin genes. Withoutlimiting the present invention in any way, each lymphoid cell undergoessomatic recombination of its germ line variable region gene segments(either V and J or V, D and J segments), depending on the particulargene arranged, in order to generate a total antigen diversity ofapproximately 10¹⁶ distinct variable region structures (note that theexpression “variable gene segment” of the TCR or immunoglobulin gene isdistinct from the expression “variant region” as hereinbefore defined).In any given lymphoid cell, such as a T cell or B cell, at least twodistinct variable region gene segment rearrangements are likely to occurdue to rearrangements involving the α, β, γ or δ chain genes of the TCRgene family and/or the heavy and light chains of the immunoglobulin genefamily. In addition to rearrangements of the VJ or VDJ segment of anygiven immunoglobulin or TCR gene, nucleotides are randomly removedand/or inserted at the junction between the segments. This leads to thegeneration of enormous diversity.

This preferred embodiment is an example of the situation where theconserved region (being the V, D or J segments) and the variant region(being the region of randomly inserted and/or deleted bases between theV, D and J segments) may either be directly linked or, where the randombase changes have extended into the V, D or J segments, the conservedportion of the V, D or J segments is linked to the variant region viathose intervening bases which have randomly mutated at the terminal endsof the V, D or J segments.

Accordingly, in a preferred embodiment the present invention is directedto an array of isolated DNA primers or derivatives or analogues thereoffor use in detecting a rearranged TCR or immunoglobulin variable genesegment, or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    portion of the 5′ end of the antisense strand of the V gene segment,    or part thereof, operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Preferably, said conserved portion of the V gene segment is a conservedportion of the F_(R3I) or F_(R3II) segment.

In another preferred embodiment the present invention is directed to anarray of isolated DNA primers or derivatives or analogues thereof foruse in detecting a rearranged TCR or immunoglobulin variable genesegment, or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    portion of the 5′ end of the antisense strand of the D gene segment,    or part thereof, operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Preferably, said recognition region is operably linked to the 3′ end ofthe nucleic acid stem region of said oligonucleotide primer.

In yet another preferred embodiment the present invention is directed toan array of isolated DNA primers or derivatives or analogues thereof foruse in detecting a rearranged TCR or immunoglobulin variable genesegment or part thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    portion of the 5′ end of the sense strand of the J gene segment, or    part thereof; operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more universally hybridising bases, or    analogues thereof, intervening said stem region and said recognition    region.

Without limiting the present invention to any one theory or mode ofaction, in order to hybridise with the target nucleic acid moleculeshereinbefore defined, the primer is designed with a stem region operablylinked to a recognition region. This design exploits the unique featureof the subject target molecules being that they comprise a variantregion sequence, which is sufficiently unique to act as a marker,located proximally to a region which is substantially conserved acrossthe class of which the target is a member. These features havefacilitated the development of primer arrays suitable for amplifying anyspecific member of such a class of molecules due to the fact that thestem region of the primer enables identification of ay molecule fallingwithin the class of interest while the recognition region enablesidentification, via either a one step or multiple step amplificationprocess, of a specific member within that class. As discussed in furtherdetail hereafter, however, the pre-synthesised primer arrays which aredesigned in accordance with the teachings provided herein can provide ahigh degree of amplification specificity and are suitable for ongoinguse as a primer source for amplification of any given target molecule ofinterest within a class of target nucleic acid molecules, without theneed to synthesise the prohibitively large numbers of primers which arecurrently required in order to achieve the same outcome.

In this regard, reference to “stem region” should be understood as areference to that portion of the subject primer which interacts with theterminal nucleotides adjoining the variant region, of the substantiallyconserved region of the target molecule. In a most preferred embodimentthese nucleotides are located at the 3′ terminal end of thesubstantially conserved region. To the extent that the substantiallyconserved region directly adjoins the variant region, the stem region isdesigned to hybridise to sufficient of the nucleic acid sequence leadingup to the point where the conserved region adjoins the variant regionsuch that the stem region would hybridise with any member of that classof molecules. This may be achieved, for example, by designing the stemregion to hybridise to the 3′ terminal four or more nucleotides, whereall members of the class comprise an identical sequence at that region.However, if there is very slight variation in sequence at that regionbetween some members, the stem region may be designed to hybridise to aconsensus sequence. Designing a suitable stem region, in terms of thenumber and sequence of the desired nucleotides, would fall well withinthe abilities of the person of skill in the art when considered in lightof the teachings provided herein. Preferably, said stem region is 5-50nucleotides in length, more preferably, 10-40 nucleotides in length, andeven more preferably, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 or 35 nucleotides in length.

That the stem region comprises a “nucleic acid interaction site” shouldbe understood as a reference to that portion of the stem region whichactually hybridises to the conserved portion of the target nucleic acidmolecule Although it is preferable, and likely, that in order to enablesynthesis of the smallest possible DNA primer the entirety of thenucleic acid component of the stem region of the primer will correspondto the nucleic acid interaction site, this may not always be the case.In some instances, it may be the case that only part of the stem regioncorresponds to the nucleic acid interaction site. This may occur, forexample, where the stem region of the primer is not linear in shape, buttakes the form of a loop or contains a 5′ tag.

As discussed hereinbefore, in some instances one or more of the terminalnucleotides adjoining the variant region, of the substantially conservedregion may themselves have undergone some degree of mutation or showsome other variation between members of the class. It should beunderstood that, in accordance with the description of the invention asprovided herein, these nucleotides are not deemed to form part of eitherthe substantially conserved region or the variant region but correspondto a region of “intervening” nucleotides. Similarly, to the extent thatthe substantially conserved region does not directly link to the variantregion in that there are one or more unrelated nucleotides positionedbetween the two regions, these are also an example of interveningnucleotides which, by definition, form part of neither the conserved orthe variant regions. In this regard, however, the design of the primersof the present invention contemplate this scenario in that the primersmay be optionally designed and synthesised such that they comprise oneor more universally hybridising nucleotides which are positioned suchthat they intervene the stem region and the recognition region of theprimer. Once more, it is well within the skill of the person in the artto determine the existence and number of any intervening nucleotides inthe context of a class of molecules to which a primer set is beingdesigned and pre-synthesised. In a preferred embodiment, the number ofuniversally hybridising bases which are built into the primer betweenthe stem region and the recognition region will correspond to the numberof intervening nucleotides.

Reference to “universally hybridising base” should be understood as areference to a molecule which can hybridise with all of guanine,cytosine, thymine, uridine or adenine. It should be understood, however,that there may exist differences in the strength of the hybridisation ofthe universally hybridising base with each of these five nucleotides.Accordingly, to the extent that some degree of hybridisation can beeffected, the “base” in issue falls within the scope of this definition.It should also be understood that the subject base may be any nucleicacid or non-nucleic acid molecule which can function in accordance withthe definition provided above. For example, there are a number of wellknown chemical modifications which can be made to the various baseswhich result in universal binding. Examples of universal bases includehypoxanthine, 5-nitroindole, 3-nitropyrrole, acyclic sugar analogues ofhypoxanthine (18) and 5-nitroindazole, phenyl C-ribonucleoside, (NucleicAcids Research, 2001, Vol. 29, No. 12 2437-2447) One may also usefunctionally equivalent means such as primers which are fully redundantat the site in issue. Accordingly, primers which include complete ornear complete redundancy for cytosine, guanine, adenine, thymine oruridine at that base location in the primer are envisaged. Preferablythe subject base is a nucleotide and even more preferably inosine orderivative, or analogue thereof.

There is therefore preferably provided an array of isolated DNA primersor derivatives or analogues thereof, for use in detecting a rearrangedTCR or immunoglobulin variable gene segment, or part thereof, saidprimers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to the 5′ end of the    antisense strand of the V gene segment, or part thereof; operably    linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more inosines, or analogues thereof,    intervening said stem region and said recognition region.

Preferably, said conserved portion of the V gene segment is a conservedportion of the F_(R3I) or F_(RII) segment.

In another preferred embodiment there is provided an array of isolatedDNA primers or derivatives or analogues thereof, for use in detecting arearranged TCR or immunoglobulin variable gene segment, or part thereof,said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to the 5′ end of the    antisense strand of the D gene segment, or part thereof; operably    linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more inosines, or analogues thereof,    intervening said stem region and said recognition region.

In still another preferred embodiment there is provided an array ofisolated DNA primers or derivatives or analogues thereof, for use indetecting a rearranged TCR or immunoglobulin variable gene segment, orpart thereof, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to the 5′ end of the sense    strand of the J gene segment, or part thereof, operably linked to-   (ii) a nucleic acid recognition region comprising at least two    nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise one or more inosines, or analogues thereof,    intervening said stem region and said recognition region.

The stem region of the primers of the present invention is operablylinked to a “nucleic acid recognition region”. By “nucleic acidrecognition region” is meant that portion of the subject primer whichcan discriminate between the members of a class of target nucleicmolecules by hybridising to the variant region of a subgroup ofmolecules within that class. Preferably, the “subgroup” of moleculescorresponds to a single target molecule of interest. However, since theprimer array of the present invention is predicated on reducing thenumber of primers which are required to be synthesised for inclusion ina pre-synthesised array by minimising the number of nucleotides whichform part of the recognition region and therefore minimising the totalnumber of nucleotide combinations (4^(n)) which are required to begenerated to form a complete primer set, it is possible that someindividual primers may hybridise with more than one member of a class ofinterest. As described hereinafter, this outcome may necessitate theapplication of an additional and subsequent amplification step utilisinga primer selected from a pre-synthesised array which is neverthelessdesigned in accordance with the teachings provided hereinbefore butwhich can function to further discriminate the multiple target classmembers which may have been amplified by an initial round ofamplification. The nucleic acid recognition region may comprise anynumber of nucleotides. The most suitable number of nucleotides is thatnumber which both provides an acceptable level of discrimination (eitherin the context of a single step or multiple consecutive stepamplification process) but minimises the number of primers required tobe pre-synthesised in order to prepare a complete array (this numberbeing 4^(n) where n is the number of nucleotides comprising the nucleicacid recognition region). This number can be routinely determined bythose of skill in the art. Specifically, the number is determined bybalancing the extra specificity resulting from an increased number ofnucleotides, as against the extra cost involved in synthesis and use.Preferred numbers of nucleotides comprising the recognition region are2, 3, 4, 5 or 6, more preferably 3 or 4 and most preferably 4.

As detailed hereinbefore, the primer array of the present inventionprovides a pre-synthesised array from which a primer can be selectedwhich will, either in a single step amplification process or aconsecutive multi-step amplification process, detect a specific targetnucleic acid molecule of interest. The target molecule is characterisedby a unique variant region, a portion of which (2, 3 or 4 nucleotides atthe 5′ terminal end of the variant region, for example) is the targetfor the primer's nucleic acid recognition region. In order to facilitatethe generation of a primer array which can be used to supply a suitableprimer to enable detection of any member of the defined class, it isnecessary that primers are synthesised which correspond to everypossible combination of adenine, guanine, cytosine and thymine/uracil.Accordingly, the number of primers which any given array comprises willequate to 4^(n) where n is the number of nucleotides comprising thenucleic acid recognition region, as follows:

Nucleotide number of primer Primer array size recognition region (numberof primers) 2 16 3 64 4 256 5 1024 6 4096

Arguably, the primer arrays of 1024 and 4096 could be regarded asprohibitively large. Accordingly, and as detailed before, the preferrednumber of nucleotides comprising the primer recognition sequence is 3 or4, most preferably 4, thereby dictating an array size of 64 or 256respectively. In this regard, reference to the primers comprising a“unique nucleic acid region recognition sequence relative to oneanother” should be understood to mean that each of the primers comprisesa unique combination of the nucleotides adenine, guanine, cytosine andthymine/uracil. However, although it is preferable that the array bedesigned such that it comprises one primer corresponding to each of thepossible nucleotide combinations (thereby envisaging arrays of 64 or 256primers, for example) the present invention nevertheless extends toarrays which may encompass multiple copies of any one or more primers orwhich do not include a primer corresponding to one or more specificnucleotide combinations. This may occur, for example, where it is knownthat a given class of target molecules does not or cannot comprise avariant region corresponding to a particular sequence. In this case itwould therefore not be necessary to synthesise primers comprising anucleic acid recognition region which is complementary to and wouldtherefore hybridise with these non-existent variant regions.

In accordance with these preferred embodiments the present inventionprovides an array of isolated DNA primers or derivatives or analoguesthereof, for use in detecting a target nucleic acid molecule which is amember of a class of nucleic acid molecules which are characterised by aspecific variant region sequence, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising three nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise at least two inosines, or analogues thereof,    intervening said stem region and said recognition region.

In another preferred embodiment there is provided an array of isolatedDNA primers or derivatives or analogues thereof, for use in detecting atarget nucleic acid molecule which is a member of a class of nucleicacid molecules which are characterised by a specific variant regionsequence, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    regions of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising four nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise at least two inosines, or analogues thereof,    intervening said stem region and said recognition region.

Still more preferably, said target nucleic acid molecule is a rearrangedTCR or immunoglobulin variable region gene, or part thereof, and saidsubstantially conserved portion is the 3′ end of the V gene segment.

The nucleic acid recognition region of the present invention is directedto a portion of the variant region of the target nucleic acid moleculeof interest. In this regard, that portion may correspond to any part ofthe variant region and does not necessarily correspond to one of theterminal ends of that region, although this would be, in the firstinstance, a preferred option. That is, in one preferred embodiment aprimer is designed in which the nucleic acid interaction site of thestem region is directed to a stretch of nucleotides at the 3′ terminalend of the substantially conserved portion of the target while thenucleic acid molecule recognition region is designed to hybridise to anucleotide stretch at the 5′ terminal end of the variant region of thetarget molecule. However, there is sometimes not an abrupt transitionbetween the substantially conserved region and the variant region, forexample as occurs in the context of TCR variable gene rearrangementswhere the 3′ terminal nucleotides of the V region can be randomlymutated during the rearrangement event. There may also be randomnucleotides inserted between the conserved region and the variantregion. In this case, the primer is designed to incorporate acorresponding number of universally hybridising molecules (for example,inosine) intervening the stem region and the recognition region. In thissituation (or even where the conserved and variant regions directlyadjoin), a given primer may nevertheless detect and amplify more thanone member of a class of target molecules. This may evidence the factthat the variation between the actual target molecule of interest andthe additional molecule(s) which are also amplified may not lie at theterminal 5′ end of the variant region. That is, the variation may, infact lie further into the variant region in the 3′ direction. If so, theprimers of the present invention can, in fact be designed to provide anadditional array, which is also not prohibitively or impracticallylarge, from which a primer can be selected to provide a still furtherlevel of discrimination of the initially amplified material. Thoseprimers, to the extent that they are utilised in the context of anadditional and consecutive amplification reaction, are herein termed“leap frog primers” or “second generation primers”. Specifically, thefurther “leap frog” primer panel is designed such that the stem regionof the leap frog primers corresponds to the stem region of the firstused primer (“first generation primer”) which is adjacent to

-   (i) intervening bases, the number of which is equal in number to the    number of universally hybridising bases which were used in the first    primer and the nature of which is determined by the nature of the    universally hybridising bases used in the first primer. In the    situation where the intervening bases in the first primer were    inosines, these intervening bases in the second primer are    preferably guanines. These intervening bases are then adjacent to:-   (ii) universally hybridising bases, the number of which is equal to    the number of nucleotides of the recognition region of the first    used primer.

Following these intervening universally hybridising bases, the primerscomprise a nucleic acid recognition region which comprises the variouscombinations of a further two or more nucleotides, preferably 3 or 4.One of these recognition region sequences will complement the nucleotidesequence immediately 3′ to the nucleotide stretch which hybridised tothe nucleic acid recognition region of the first used primer. The use ofa suitably selected primer from this additional array provides anadditional level of discrimination when used to probe and amplify thenucleic acid material amplified via use of the first primer. This allowsone to screen for and amplify a target nucleic acid molecule utilisingprimers selected from two primer arrays, thereby providing a very highdegree of specificity, each consisting of 256 primers in the preferredembodiment—thereby requiring a total of only 512 primers to have beengenerated. These arrays of primers however, are suitable for repeateduse as a source of specific primers in the context of detecting anytarget nucleic acid molecule which falls within the class of moleculesagainst which the arrays were generated. To the extent that thedetection of a particular target sequence requires the use of a 2-stepconsecutive amplification process which utilises a primer directed to aninitial four nucleotide stretch of the variant region followed by use ofa primer directed to the next four nucleotide stretch (in the 3′direction), one would arguably have required a primer complementary tothe sequence of the first 8 nucleotide stretch of the variant region ifthe detection was to have been performed in a single step. To generate apre-synthesised set of primers which hybridise at the level of 8nucleotides and from which on appropriate primer could have beenselected would have require the generation of an array comprising 4⁸(65,536) primers—this being an entirely prohibitive number tosynthesise, both in terms of practicality and cost.

It should be understood, that the use of leap frog primers is notnecessarily limited to second or third round amplification processes inthat they may also be utilised for initial amplification processes iftheir design may be suitable for a first round amplification process.

In the context of the earlier definitions provided in relation to thatportion of the variant region to which the nucleic acid recognitionregion of the primer is directed, the above described primer set whichis suitable for use subsequently to an initial amplification step is anexample of a primer in which the nucleic acid recognition region isdirected to a sequence of nucleotides which are not located at theterminal end of the variant region where it adjoins either the conservedregion or any intervening nucleotides which may exist between thevariant region and the conserved region. Rather it is directed to asequence of nucleotides downstream of the 3′ terminal end of the variantregion. In the context of these primers, which are primarily designedfor use subsequently to an initial amplification step, it should beunderstood that the additional universally hybridising bases which areinserted into the primer for the purpose of hybridising to thenucleotides which hybridised to the nucleic acid recognition region ofthe first used primer fall within the definition of universallyhybridising molecules “intervening said stem region and said recognitionregion” as hereinbefore discussed.

It should be understood that the oligonucleotide of the presentinvention should not be limited to the specific structure exemplifiedherein (being a linear, single-stranded molecule) but may extend to anysuitable structural configuration which achieves the functionalobjectives detailed herein. For example, it may be desirable that all orpart of the oligonucleotide is double stranded, comprises a loopedregion, such as a hairpin bend or takes the form of an open circleconformation, that is, where the nucleotide primer is substantiallycircular in shape but its terminal regions do not connect.

Reference to a “nucleic acid” should be understood as a reference toboth deoxyribonucleic acid, ribonucleic acid, a combination of both orderivatives or analogues thereof. The nucleic acid molecules utilised inthe present invention may be of any origin including naturally occurring(for example a biological sample may be utilised), recombinantlyproduced or synthetically produced. Preferably, said nucleic acid isdeoxyribonucleic acid.

Facilitating the interaction of the nucleic acid probe with the targetnucleic acid sequence may be performed by any suitable method. Thosemethods will be known to those skilled in the art.

In accordance with these preferred embodiments the present inventionprovides an array of isolated DNA primers or derivatives or analoguesthereof, for use in detecting a target nucleic acid molecule which is amember of a class of nucleic acid molecules which are characterised by aspecific variant region sequence, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    region of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising three nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise at least two inosines, or analogues thereof,    intervening said stem region and said recognition region.

In another preferred embodiment there is provided an array of isolatedDNA primers or derivatives or analogues thereof, for use in detecting atarget nucleic acid molecule which is a member of a class of nucleicacid molecules which are characterised by a specific variant regionsequence, said primers comprising:

-   (i) a nucleic acid stem region, which stem region comprises a    nucleic acid interaction site directed to a substantially conserved    regions of the class of which said target nucleic acid molecule is a    member, or part thereof, and which substantially conserved region is    located proximally to a variant region; operably linked to-   (ii) a nucleic acid recognition region comprising four nucleotides    wherein said primers comprise unique nucleic acid recognition region    sequences relative to one another and wherein said primers    optionally comprise at least two inosines, or analogues thereof,    intervening said stem region and said recognition region.

Reference to the nucleic acid stem region being “operably linked” to thenucleic acid recognition region should be understood as a reference tothese regions being linked such that the functional objective, beinghybridisation of the oligonucleotide to a target nucleic acid moleculeand, optionally, amplification therefrom can be achieved. In thisregard, and as detailed hereinbefore, it may be necessary that the stemregion and the nucleic acid recognition region are linked via one ormore universally hybridising bases. This can be necessitated by thestructure of the target molecule in terms of the position of the portionof the conserved region which is the target of the stem region relativeto the portion of the variant region which is the target of therecognition region, in terms of intervening nucleotides. Accordingly,where it is necessary to design an oligonucleotide of the presentinvention with a region of intervening universally hybridisingnucleotides, it should be understood that the stem region and therecognition region are nevertheless operably linked. In terms of themeans by which these regions are linked and, further, the means by whichthe subject oligonucleotide binds to its target molecule, thesecorrespond to various types of interactions. In this regard, referenceto “interaction” should be understood as a reference to any form ofinteraction such as hybridisation between complementary nucleotide basepairs or some other form of interaction such as the formation of bondsbetween any nucleic or non-nucleic acid portion of the primer moleculewith any nucleic acid or non-nucleic acid portion of the targetmolecule. This type of interaction may occur via the formation of bondssuch as, but not limited to, covalent bonds, hydrogen bonds, van derWals forces or any other mechanism of interaction. Preferably, to theextent that the interaction occurs between the primer and a targetmolecule, said interaction is hybridisation between complementarynucleotide base pairs. All references herein to “hybridisation” betweentwo nucleic acid molecules should be understood to encompass any form ofinteraction between said molecules. In order to facilitate thisinteraction, it is preferable that the target nucleic acid molecules arerendered partially or fully single stranded for a time and underconditions sufficient for hybridisation with a primer to occur. To theextent that the interaction occurs between the different regions of theprimer molecule, these interactions will preferably occur directlybetween adjacent nucleotides but may also occur between any non-nucleicacid components which may form part of these regions. To the extent thatthe interaction does occur directly between the nucleotides, theseinteractions preferably take the form of covalent bonds which correspondto 3′, 5′ phosphodiester linkages.

As would be appreciated, in order to design the oligonucleotides of thepresent invention, it is necessary to first establish that the targetnucleic acid molecule of interest is characterised by a substantiallyconserved region which is located proximally to a variant region and,secondly, to determine the nucleotide sequence of the conserved regionto enable the design of a suitable primer array. In order to select asuitable primer for use, one does require some sequence information inrelation to the variant region of the target of interest. Methods fordoing so are routine and would be well known to those of skill in theart.

The development of the oligonucleotides of the present invention nowprovides a means of efficiently facilitating the routine screening ofpopulations of nucleic acid molecules for the presence of a targetnucleic acid molecule which is characterised by a specific variantregion located proximally to a substantially conserved region. Asdetailed hereinbefore, this efficiency is due to the determination thattarget nucleic acid molecules of this type lend themselves to thegeneration of a relatively small number of individual primers which, inthe form of an array, can achieve the hybridisation to and amplificationof a target molecule of interest, within the class of molecule to whichthe primer was generated, exhibiting any possible variant regionsequence. As detailed hereinbefore, this can be achieved through eithera single step or consecutive multiple step amplification processutilising primers selected from arrays which have been designedaccording to the present invention. Accordingly, the present inventionis particularly useful in the context of applications such as thedetection, identification, quantitation and/or typing of specificgenetic sequences found in biological or environmental samples such asmolecular sequences of human, animal, plant, parasite, bacterial orviral origin. This includes, but is not limited to, allelicdiscrimination of genes, identification and/or isolation of geneticvariants or mutants (for example, for the purpose of predicting patientdrug responses), identification of molecules expressing specific singlenucleic polymorphisms (SNPs) or for the identification and/or isolationof particular microorganism strains or the detection of mutationsthereof.

An example of how such pre-synthesised primer panels could be used comesfrom amplification of immunoglobulin or T cell rearrangements.Rearranged immunoglobulin or T cell receptor genes are often used asmolecular markers to detect low numbers of cancerous lymphocytes, eg inleukaemia, lymphoma or myeloma. Although there are some minorvariations, each individual cancer in an individual patient is a clone,deriving from a single lymphocyte which has become malignant, and allcells of the tumour bear the same rearrangement. In attempting to detectlow numbers of leukaemic cells, a standard approach is to use therearranged immunoglobulin or T cell receptor gene as a molecular markerand to attempt to specifically amplify and quantify the specificrearrangement. In order to do this, it is necessary to use primers whichprovide a greater or lesser degree of specific amplification of therearrangement of the malignant clone and which do not amplify otherrandom rearrangements derived from non-malignant cells. By sequencingthe particular rearrangement at diagnosis, one can select and synthesisethe particular primer or primers which bind to and amplify therearrangement from the malignant clone and which show little or nobinding to other rearrangements. However this involves separatesynthesis and testing for each patient, which in aggregate becomestime-consuming and expensive when many patients are being studied.

In contrast to the above situation, in which it is desired to monitor asingle clone bearing a molecular marker of a sequence which has beendetermined and which indicates the member of the oligonucleotide panelwhich should be chosen and used for amplification, it is sometimesdesired to study a cell population which contains many clones, each ofwhich is defined by a different DNA sequence. If a hypervariable regionis responsible for much of the DNA sequence differences, as obtains forthe N regions of the rearranged immunoglobulin or T cell receptor genes,then it will be possible to define subpopulations of cells on the basisof differences in the DNA sequences at the 5′ end of the hypervariableregions. The sizes of these various subpopulations can be determined byperforming multiple parallel nucleic aid amplifications with eachdifferent amplification reaction containing a different oligonucleotidefrom the panel. This enables an assessment of the repertoire ofsubpopulations within either a malignant or a non-malignant populationof cells and it also enables identification of one or moresubpopulations of cells which, owing to their absolute or changing size,it may be desired to follow subsequently.

These methods, which are now enabled by the development of theoligonucleotides disclosed herein, therefore have broad applicationincluding but not limited to:

-   (i) providing a means of monitoring the progression of a clonal    population of cells in a subject. This is most likely to occur in    the context of monitoring a patient in terms of the progression of a    disease state or non-disease state which is characterised by the    clonal expansion of a population of cells. For example, there is    significant potential for the application of the method of the    present invention in terms of patients suffering from malignant and    non-malignant neoplasias. However, there may also be potential to    apply the present invention in the context of patients suffering    various forms of immunodeficiency, where one may seek to screen for    the nature of specific immune cell expansion which can be mounted by    that individual's immune system.-   (ii) a means of diagnosing a disease condition where, for example,    either the appearance or loss of a gene expressing a specific    variant sequence (i.e. a mutant gene) either per se or relative to a    certain threshold levels correlates to the onset of a particular    condition. Such mutations may be congenital or they may have been    acquired by virtue of exposure to an adverse environment factor    (e.g. radiation). In addition to diagnosis, one can monitor the    progress of such a disease condition.-   (iii) diagnosing the presence of and/or monitoring levels of    infection by a particular strain or variant of a microorganism (for    example, an HIV or influenza variant), where that microorganism is    characterised by a proximally located variant region/conserved    region junction as hereinbefore defined. Also provided is the    clinical diagnosis of a disease state or other condition which is    induced by or related to the occurrence of an infection with a    specific genetic form of a microorganism, such as a genetically    unique bacterium, virus or parasite.-   (iv) the conserved region-variant region junction sequences provide    a means of marking a population of cells. For example, once these    sequences have been identified, one can routinely screen populations    of cells in order to identify (either qualitative and/or    quantitatively) the existence of the population of cells expressing    that specific marker. The method of the present invention therefore    provides a relatively routine means of characterising a clonal cell    population and provides for ongoing detection/monitoring    applications without the need to conduct elaborate genetic analyses.

Accordingly, another aspect of the present invention is directed to amethod of identifying a target nucleic acid molecule, which molecule isa member of a class of nucleic acid molecules characterised by aspecific variable region sequence, in a sample said method comprising

-   (i) contacting said sample with an oligonucleotide as hereinbefore    defined for a time and under conditions sufficient to facilitate    interaction of said oligonucleotide with said target nucleic acid    molecule;-   (ii) amplifying said nucleic acid target; and-   (iii) optionally consecutively repeating said amplification steps    utilising the nucleic acid material amplified in the previous step    together with a leap frog oligonucleotide; and-   (iv) detecting said amplified product.

Preferably, said oligonucleotides are primers and, even more preferably,DNA primers. Yet more preferably, said nucleotide acid recognitionregion comprises three or four nucleotides and said universallyhybridising base is inosine.

In a most preferred embodiment, said target nucleic acid molecule is arearranged TCR or immunoglobulin variable region gene, or part thereof,and said substantially conserved portion is the 3′ end of the V genesegment.

As detailed hereinbefore reference to a “leap frog” or “secondgeneration” oligonucleotide should be understood as a reference to thepopulation of oligonucleotides which have been designed to provide afurther level of discrimination to that afforded utilising an initialoligonucleotide. Specifically, the oligonucleotides which are initiallyutilised are designed, preferably, with the minimal number ofnucleotides directed to the conserved region and the variant regionrequired to either select or at least enrich for the target nucleic acidpopulation. To the extent that the target nucleic acid population hasbeen enriched, a further panel of oligonucleotides can be designedcomprising:

-   (i) a stem region;-   (ii) a region of specific bases which hybridises to the bases in the    amplified target corresponding to the universally hybridising bases    of the first primer;-   (iii) universally hybridising bases which correspond to the bases of    recognition sequence of the primer and-   (iv) a recognition sequence.

The “leap frog” oligonucleotide is then designed with a recognitionregion directed to the nucleotide sequence 3′ to the sequence recognisedby the recognition region of the first oligonucleotide.

Methods for achieving primer directed amplification are well known tothose of skill in the art. In a preferred method, said amplification ispolymerase chain reaction, NASBA or strand displacement amplification.

Reference to a “sample” should be understood as a reference to either abiological or a non-biological sample. Examples of non-biologicalsamples includes, for example, the nucleic acid products ofsynthetically produced nucleic acid populations. Reference to a“biological sample” should be understood as a reference to any sample ofbiological material derived from an animal, plant or microorganism(including cultures of microorganism) such as, but not limited to,cellular material, blood, mucus, faeces, urine, tissue biopsy specimens,fluid which has been introduced into the body of an animal andsubsequently removed (such as, for example, the saline solutionextracted from the lung following lung lavage or the solution retrievedfrom an enema wash), plant material or plant propagation material suchas seeds or flowers or a microorganism colony. The biological samplewhich is tested according to the method of the present invention may betested directly or may require some form of treatment prior to testing.For example, a biopsy sample may require homogenisation prior totesting. For example, a biopsy sample may require homogenisation priorto testing or it may require sectioning for in situ testing. Further, tothe extent that the biological sample is not in liquid form, (if suchform is required for testing) it may require the addition of a reagent,such as a buffer, to mobilise the sample.

To the extent that the target molecule is present in a biologicalsample, the biological sample may be directly tested or else all or someof the nucleic acid material present in the biological sample may beisolated prior to testing. It is within the scope of the presentinvention for the target nucleic acid molecule to be pre-treated priorto testing, for example, inactivation of live virus or being run on agel. It should also be understood that the biological sample may befreshly harvested or it may have been stored (for example by freezing)prior to testing or otherwise treated prior to testing (such as byundergoing culturing).

Reference to “contacting” the sample with the primer should beunderstood as a reference to facilitating the mixing of the primer withthe sample such that interaction (for example, hybridisation) can occur.Means of achieving this objective would be well known to those of skillin the art.

The choice of what type of sample is most suitable for testing inaccordance with the method disclosed herein will be dependent on thenature of the situation, such as the nature of the condition beingmonitored. For example, in a preferred embodiment a neoplastic conditionis the subject of analysis. If the neoplastic condition is a lymphoidleukaemia, a blood sample, lymph fluid sample or bone marrow aspiratewould likely provide a suitable testing sample. Where the neoplasticcondition is a lymphoma, a lymph node biopsy or a blood or marrow samplewould likely provide a suitable source of tissue for testing.Consideration would also be required as to whether one is monitoring theoriginal source of the neoplastic cells or whether the presence ofmetastases or other forms of spreading of the neoplasia from the pointof origin is to be monitored. In this regard, it may be desirable toharvest and test a number of different samples from any one mammal.Choosing an appropriate sample for any given detection scenario wouldfall within the skills of the person of ordinary skill in the art.

The term “mammal” to the extent that it is used herein includes humans,primates, livestock animals (e.g. horses, cattle, sheep, pigs, donkeys),laboratory test animals (e.g. mice, rats, rabbits, guinea pigs),companion animals (e.g. dogs, cats) and captive wild animals (e.g.kangaroos, deer, foxes). Preferably, the mammal is a human or alaboratory test animal. Even more preferably the mammal is a human.

The method of this aspect of the present invention provides a means forboth detecting the presence of a target nucleic acid molecule ofinterest and, optionally, quantifying and/or isolating that target.Accordingly, one is provided with means of either enriching or purifyinga target nucleic acid population of interest for any purpose, such asfurther analysis of the target.

It should be understood that the execution of the method of the presentinvention is not intended to be limited to the specific means detailedherein since the design and application of such means would be wellwithin the skill of the person of skill in the art.

Another aspect of the present invention provides a method of detectingand/or monitoring a clonal population of cells in a mammal, which clonalcells are characterised by a target nucleic acid molecule which is amember of a class of nucleic acid molecules characterised by a specificvariant region sequence, said method comprising:

-   (i) contacting the nucleic acid material of a biological sample    derived from a mammal with an oligonucleotide as hereinbefore    defined for a time and under conditions sufficient to facilitate    interaction of said oligonucleotide with said target nucleic acid    molecule;-   (ii) amplifying said nucleic acid target;-   (iii) optionally consecutively repeating said amplification steps    utilising the nucleic acid material amplified in the preceding step    together with a leap frog oligonucleotide, and-   (iv) detecting said amplified product.

Preferably, said oligonucleotides are primers and, even more preferably,DNA primers. Yet more preferably, said nucleotide acid recognitionregion comprises three or four nucleotides and said universallyhybridising base is inosine.

In a most preferred embodiment, said target nucleic acid molecule is arearranged TCR or immunoglobulin variable region gene, or part thereof,and said substantially conserved portion is the 3′ end of the V genesegment.

Reference to “cells” should be understood as a reference to all forms ofcells from any species and to mutants or variants thereof. Withoutlimiting the present invention to any one theory or mode of action, acell may constitute an organism (in the case of unicellular organisms)or it may be a subunit of a multicellular organism in which individualcells may be more or less specialised (differentiated) for particularfunctions. All living organisms are composed of one or more cells. Thesubject cell may form part of the biological sample which is the subjectof testing in a syngeneic, allogeneic or xenogeneic context. A syngeneicprocess means that the clonal cell population and the biological samplewithin which that clonal population exists share the same MHC genotype.This will most likely be the case where one is screening for theexistence of a neoplasia in an individual, for example. An “allogeneic”process is where the subject clonal population in fact expresses adifferent MHC to that of the individual from which the biological sampleis harvested. This may occur, for example, where one is screening forthe proliferation of a transplanted donor cell population (such as animmunocompetent bone marrow transplant) in the context of a conditionsuch as graft versus host disease. A “xenogeneic” process is where thesubject clonal cells are of an entirely different species to that of thesubject from which the biological sample is derived. This may occur, forexample, where a potentially neoplastic donor population is derived fromxenogeneic transplant.

“Variants” of the subject cells include, but are not limited to, cellsexhibiting some but not all of the morphological or phenotypic featuresor functional activities of the cell of which it is a variant. “Mutants”includes, but is not limited to, cells which have been naturally ornon-naturally modified such as cells which are genetically modified.

By “clonal” is meant that the subject population of cells has derivedfrom a common cellular origin. For example, a population of neoplasticcells is derived from a single cell which has undergone transformationat a particular stage of differentiation. In this regard, a neoplasticcell which undergoes further nuclear rearrangement or mutation toproduce a genetically distinct population of neoplastic cells is also a“clonal” population of cells, albeit a distinct clonal population ofcells. In another example, a T or B lymphocyte which expands in responseto an acute or chronic infection or immune stimulation is also a“clonal” population of cells within the definition provided herewith. Inyet another example, the clonal population of cells is a clonalmicroorganism population, such as a drug resistant clone which hasarisen within a larger microorganismal population. Preferably, thesubject clonal population of cells is a neoplastic population of cellsor a clonal immune cell population.

Preferably said clonal cells are a population of clonal lymphoid cells.

It should be understood that reference to “lymphoid cell” is a referenceto any cell which has rearranged at least one germ line set ofimmunoglobulin or TCR variable region gene segments. The immunoglobulinvariable region encoding genomic DNA which may be rearranged includesthe variable regions associated with the heavy chain or the K or X lightchain while the TCR chain variable region encoding genomic DNA which maybe rearranged include the α, β, γ and δ chains. In this regard, a cellshould be understood to fall within the scope of the “lymphoid cell”definition provided the cell has rearranged the variable region encodingDNA of at least one immunoglobulin or TCR gene segment region. It is notnecessary that the cell is also transcribing and translating therearranged DNA. In this regard, “lymphoid cell” includes within itsscope, but is in no way limited to, immature T and B cells which haverearranged the TCR or immunoglobulin variable region gene segments butwhich are not yet expressing the rearranged chain (such as TCR⁻thymocytes) or which have not yet rearranged both chains of their TCR orimmunoglobulin variable region gene segments. This definition furtherextends to lymphoid-like cells which have undergone at least some TCR orimmunoglobulin variable region rearrangement but which cell may nototherwise exhibit all the phenotypic or functional characteristicstraditionally associated with a mature T cell or B cell. Accordingly,the method of the present invention can be used to monitor neoplasias ofcells including, but not limited to, lymphoid cells at anydifferentiative stage of development, activated lymphoid cells ornon-lymphoid/lymphoid-like cells provided that rearrangement of at leastpart of one variable region gene region has occurred. It can also beused to monitor the clonal expansion which occurs in response to aspecific antigen.

It should also be understood that although it is preferable that therearrangement of at least one variable region gene region has beencompleted, the method of the present invention is neverthelessapplicable to monitoring neoplastic cells which exhibit only partialrearrangement. For example, a B cell which has only undergone the DJrecombination event is a cell which has undergone only partialrearrangement. Complete rearrangement will not be achieved until the DJrecombination segment has further recombined with a V segment. Themethod of the present invention can therefore be designed to detect thepartial or complete variable region rearrangement of one TCR orimmunoglobulin chain utilising a reference molecule complementary tothis marker sequence or, for example, if greater specificity is requiredand the neoplastic cell has rearranged the variable region of both TCRor immunoglobulin chains, primer molecules directed to both forms ofrearrangement can be utilised.

Reference to a “neoplastic cell” should be understood as a reference toa cell exhibiting abnormal “growth”. The term “growth” should beunderstood in its broadest sense and includes reference toproliferation. In this regard, an example of abnormal cell growth is theuncontrolled proliferation of a cell. The uncontrolled proliferation ofa lymphoid cell may lead to a population of cells which take the form ofeither a solid tumour or a single cell suspension (such as is observed,for example, in the blood of a leukemic patient). A neoplastic cell maybe a benign cell or a malignant cell. In a preferred embodiment, theneoplastic cell is a malignant cell. In this regard, reference to a“neoplastic condition” is a reference to the existence of neoplasticcells in the subject mammal. Although “neoplastic lymphoid condition”includes reference to disease conditions which are characterised byreference to the presence of abnormally high numbers of neoplastic cellssuch as occurs in leukemias, lymphomas and myelomas, this phrase shouldalso be understood to include reference to the circumstance where thenumber of neoplastic cells found in a mammal falls below the thresholdwhich is usually regarded as demarcating the shift of a mammal from anevident disease state to a remission state or vice versa (the cellnumber which is present during remission is often referred to as the“minimal residual disease”). Still further, even where the number ofneoplastic cells present in a mammal falls below the thresholddetectable by the screening methods utilised prior to the advent of thepresent invention, the mammal is nevertheless regarded as exhibiting a“neoplastic condition”.

As detailed hereinbefore, the development of the primers of the presentinvention have now facilitated the development of diagnostic andmonitoring applications which are based on achieving high use specificnucleic acid discrimination utilising one or more arrays of relativelymodest numbers of presynthesised oligonucleotide primers. This haswidespread application in, inter alia, disease monitoring, diagnosis andprognosis, genetic profiling and the detection of specificmicroorganisms infections.

Accordingly, still another aspect of the present invention is directedto a method for diagnosis of the onset of or a predisposition to theonset of a disease condition or for monitoring or prognosing theprogression of a disease condition in a mammal, which condition ischaracterised by the presence or change in the level of a target nucleicacid molecule, or clonal cell population characterised by a targetnucleic acid molecule, which molecule is a member of a class nucleicacid molecule characterised by a specific variant region sequence, saidmethod comprising:

-   (i) contacting a sample derived from said mammal with an    oligonucleotide as hereinbefore defined, for a time and under    conditions sufficient to facilitate interaction of said    oligonucleotide with said target nucleic acid molecule;-   (ii) amplifying said nucleic acid target;-   (iii) optionally consecutively repeating said amplification steps    utilising the nucleic acid material amplified in the preceding step    together with a leap frog oligonucleotide; and-   (iv) detecting said amplified product.

Disease conditions suitable for analysis in this regard are any lymphoidmalignancies such as acute lymphoblastic leukaemia, chronic lymphocyticleukaemia, non-Hodgkin's lymphoma and myeloma. Monitoring of minimalresidual disease is of importance in all of these conditions. Othersituations in which this method is applicable include monitoring ofbacterial or viral infections, particularly those in which there is agreat deal of genetic variation. Human or animal retroviral infectionssuch as HIV are just one example.

With respect to this aspect of the present invention, reference to“monitoring” should be understood as a reference to testing the subjectfor the presence or level of the subject clonal population of cellsafter initial diagnosis of the existence of said population.“Monitoring” includes reference to conducting both isolated one offtests or a series of tests over a period of days, weeks, months oryears. The tests may be conducted for any number of reasons including,but not limited to, predicting the likelihood that a mammal which is inremission will relapse, monitoring the effectiveness of a treatmentprotocol, checking the status of a patient who is in remission,monitoring the progress of a condition prior to or subsequently to theapplication of a treatment regime, in order to assist in reaching adecision with respect to suitable treatment or in order to test newforms of treatment. The method of the present invention is thereforeuseful as both a clinical tool and a research tool.

Preferably, said condition is a neoplasia and even more preferably alymphoid neoplasia.

Yet another aspect of the present invention is directed to a kit forfacilitating the identification of a target nucleic acid molecule, saidkit comprising compartments adapted to contain any one or more of theoligonucleotide primers as hereinbefore defined, reagents useful forfacilitating interaction of said primer with the target nucleic acidmolecule and reagents useful for enabling said interaction to result inamplification of said nucleic acid target. Further compartments may alsobe included, for example, to receive biological or non-biologicalsamples.

Further features of the present invention are more fully described inthe following non-limiting examples.

EXAMPLE 1

Study of DNA samples from 5 patients with ALL. The effect of inosinenumber was studied by using primers 31 bases in length and, proceedingfrom the 3′ to 5′ end, having: 3 bases of perfect match (to the first 3variable bases of the N region); 0,2,4 or 6 inosines and; 28, 26, 24 or22 bases of perfect match. The effect of annealing temperature was alsostudied. The end-point was the amplification achieved by 20 cycles ofPCR. Amplification fell below 10⁴ when primers containing 6 inosineswere used. In this experiment, temperature had no effect but an effectwas seen in some other experiments. The final conditions when usingprimers directed at 3 variable bases were: primers containing 4 inosinesat an annealing temperature of 43 Celsius. Another experiment showedthat a primer directed towards 4 variable bases was tolerant to 6 or 8inosines at 43 Celsius (FIG. 1).

EXAMPLE 2

Results of detection of low numbers of leukemic cells in 7 experimentsin which the leukemic cells were mixed in various proportions withnormal peripheral blood cell. Specific amplification of the leukemiccells was achieved by 3 sequential PCRs, the second of which involved aprimer containing 4 inosines followed by 3 bases at the 3′ end whichmatched the first 3 bases of the N region. There is excellentcorrelation between the observed results and the theoretical results asexpected from the mixtures that were made (FIG. 2).

EXAMPLE 3

ino- final Patient/Expt sines 3′ bases Site Result 1 Result 2 393/03 3944 TTG A1 <1.2 × 10 −6 3.7 × 10 −5 395 A2 <1.2 × 10 −6 4.6 × 10 −5 396 A33.71 × 10 −6 <2.2 × 10 −6 397 B1 9.7 × 10 −5 1.8 × 10 −5 398 B2 <1.2 ×10 −6 5.04 × 10 −5 399/03 4 CGG 400 A1 8.1 × 10 −4 3.3 × 10 −4 401 A22.43 × 10 −4 1.22 × 10 −4 402 A3 4.47 × 10 −4 3.89 × 10 −4 403 B1 2.53 ×10 −4 1.46 × 10 −4 404 B2 3.4 × 10 −4 3.3 × 10 −4 405/03 4 TTT 406 A11.3 × 10 −6 1.84 × 10 −5 407 A2 9.6 × 10 −6 <2.2 × 10 −7 408 A3 6.52 ×10 −6 <3.3 × 10 −7 409 B1 3.1 × 10 −5 2.9 × 10 −5 410 B2 1.2 × 10 −5 7.8× 10 −6 411/03 4 CGT 412 A1 3.22 × 10 −7 3.07 × 10 −7 413 A2 <4 × 10 −7<4 × 10 −7 414 A3 <4 × 10 −7 <4 × 10 −7 415 B1 <4 × 10 −7 <4 × 10 −7 416B2 <4 × 10 −7 <4 × 10 −7 417/03 4 TCAA 418 A1 1.02 × 10 −2 4.1 × 10 −3419 A2 1.05 × 10 −2 4.46 × 10 −3 420 A3 3.51 × 10 −2 1.3 × 10 −2 421 B11.41 × 10 −2 6.3 × 10 −2 422 B2 1.04 × 10 −2 1.20 × 10 −2 102/04 6 TTAA90 A1 <2.14 × 10 −6 <3.02 × 10 −7 91 A2 1.76 × 10 −5 <2.19 × 10 −7 92 A3<2.4 × 10 −6 <2.84 × 10 −7 93 A4 <2.5 × 10 −6 <2.2 × 10 −7 94 A5 <1.5 ×10 −6 <2.2 × 10 −7 228/04 6 CCGA 299 A1 3.9 × 10 −5 1.56E−07 230 A2 1.8× 10 −5 1.66E−06 231 A3 5.5 × 10 −5 1.85E−06 232 B1 5.28 × 10 −55.72E−06 233 B2 1.39 × 10 −5 9.98E−06 179/04 6 TGGA 180 A1 2.10E−04 181A2 1.85E−04 182 A3 8.49E−05 183 B1 1.99E−04 6.81E−05 184 B2 8.61E−055.40E−06 222/04 6 AAAA 223 A1 3.91E−05 2.49E−05 224 A2 1.73E−05 2.26E−05225 A3 3.24E−05 1.57E−05 226 B1 7.49E−06 4.86E−06 227 B2 2.58E−053.94E−05 216/04 6 GATA 217 A1 7.76E−04 218 A2 6.24E−04 219 A3 2.75E−04220 B1 2.05E−04 221 B2 4.70E−04

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

1. An array of isolated nucleic acid molecules or derivatives oranalogues thereof, for use in detecting a target nucleic acid moleculewhich is a member of a class of nucleic acid molecules and which ischaracterised by a specific variant region, said nucleic acid moleculescomprising: (i) a nucleic acid stem region, which stem region comprisesa nucleic acid interaction site directed to a substantially conservedregion of the class of which said target nucleic acid molecule is amember, or part thereof and which substantially conserved region islocated proximally to a variant region; operably linked to (ii) anucleic acid recognition region comprising at least two nucleotideswherein said nucleic acid molecules comprise unique nucleic acidrecognition region sequences relative to one another and wherein saidnucleic acid molecules optionally comprise one or more universallyhybridising bases, or analogues thereof, intervening said stem regionand said recognition region.
 2. An isolated nucleic acid molecule orderivative or analogue thereof, for use in detecting a target nucleicacid molecule which is a member of a class of nucleic acid molecules andwhich is characterised by a specific variant region, said nucleic acidmolecule comprising: (i) a nucleic acid stem region, which stem regioncomprises a nucleic acid interaction site directed to a substantiallyconserved region of the class of which said target nucleic acid moleculeis a member, or part thereof and which substantially conserved region islocated proximally to a variant region; operably linked to (ii) anucleic acid recognition region comprising at least two nucleotideswherein said nucleic acid molecule optionally comprises one or moreuniversally hybridising bases, or analogues thereof, intervening saidstem region and said recognition region.
 3. The array according to claim1 or molecule according to claim 2 wherein said recognition region isoperably linked to the 3′ end of the nucleic acid stem region of saidisolated nucleic acid molecule.
 4. The array according to claim 1 or 3or molecule according to claim 2 or 3 wherein said class of nucleic acidmolecules is the rearranged genomic immunoglobulin genes.
 5. The arrayor molecule according to claim 4 wherein said rearranged genomicimmunoglobulin gene is the heavy chain gene.
 6. The array or moleculeaccording to claim 4 wherein said rearranged genomic immunoglobulin geneis the light chain gene.
 7. The array according to claim 1 or 3 ormolecule according to claim 2 or 3 wherein said class of nucleic acidmolecules is the rearranged genomic T cell receptor genes.
 8. The arrayor molecule according to claim 7 wherein said rearranged genomic T cellreceptor gene is the α chain gene.
 9. The array or molecule according toclaim 7 wherein said rearranged genomic T cell receptor gene is the βchain gene.
 10. The array or molecule according to claim 7 wherein saidrearranged genomic T cell receptor gene is the y chain gene.
 11. Thearray or molecule according to claim 7 wherein said rearranged genomic Tcell receptor gene is the 8 chain gene.
 12. The array or moleculeaccording to any one of claims 4 to 11 wherein said nucleic acidinteraction site is directed to a substantially conserved portion of the5′ end of the antisense strand of the V gene segment.
 13. The array ormolecule according to claim 12 wherein said conserved portion of the Vgene segment is a conserved portion of the F_(R3I) or F_(R3II) segment.14. The array or molecule according to any one of claims 4, 5, 7, 9, 10or 11 wherein said nucleic acid interaction site is directed to asubstantially conserved portion of the 5′ end of the antisense strand ofthe D gene segment.
 15. The array or molecule according to any one ofclaims 4 to 11 wherein said nucleic acid interaction site is directed toa substantially conserved portion of the 5′ end of the sense strand ofthe J gene segment.
 16. The array or molecule according to any one ofclaims 4, 5, 7, 9, 10 or 11 wherein said nucleic acid interaction siteis directed to a substantially conserved portion of the 5′ end of thesense strand of the D gene segment.
 17. The array or molecule accordingto any one of claims 1 to 16 wherein said stem region is from 5 to 50nucleotides in length.
 18. The array or molecule according to claim 17wherein said stem region is from 10 to 40 nucleotides in length.
 19. Thearray or molecule according to claim 18 wherein said stem region is from15 to 35 nucleotides in length.
 20. The array or molecule according toclaim 19 wherein said stem region is from 20 to 35 nucleotides inlength.
 21. The array or molecule according to claim 20 wherein saidstem region is 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34 or 35 nucleotides in length.
 22. The array or molecule according toany one of claims 17 to 21 wherein said nucleic acid recognition regionis 2, 3, 4, 5 or 6 nucleotides in length.
 23. The array or moleculeaccording to claim 22 wherein said nucleic acid recognition region is 3nucleotides in length.
 24. The array or molecule according to claim 22wherein said nucleic acid recognition region is 4 nucleotides in length.25. The array or molecule according to any one of claims 1 to 24 whereinsaid isolated nucleic acid molecule is an isolated oligonucleotide. 26.The array or molecule according to claim 25 wherein said oligonucleotideis an oligonucleotide primer.
 27. The array or molecule according toclaim 26 wherein said oligonucleotide primer is DNA.
 28. The array ormolecule according to claim 27 wherein said universally hybridising baseis inosine.
 29. The array or molecule according to claim 28 wherein saidisolated nucleic acid molecule comprises at least two inosines.
 30. Asecond generation isolated nucleic acid molecule or derivative oranalogue thereof, for use in detecting a target nucleic acid moleculewhich is a member of a class of nucleic acid molecules and which ischaracterised by a specific variant region, said nucleic acid moleculecomprising: (i) a stem region which corresponds to the stem region ofthe first generation nucleic acid molecule of any one of claims 1 to 28;operably linked to (ii) an intervening base region, which interveningbase region is directed to the universally hybridising bases of thefirst generation nucleic acid molecule and which intervening base regionis operably linked to (iii) a region of universally hybridising basesdirected to the nucleic acid recognition region of said first generationnucleic acid molecule and which region of universally hybridising basesis operably linked to (iv) a nucleic acid recognition region comprisingat least two nucleotides directed to the nucleotide sequence 3′ to thesequence to which the recognition region of the first generation nucleicacid molecule is directed.
 31. An array of second generation nucleicacid molecules according to claim 30 wherein said nucleic acid moleculescomprise unique nucleic acid recognition sequences relative to oneanother.
 32. The array according to any one of claims 1, 3 to 29 or 31wherein said array comprises two or more nucleic acid moleculescomprising unique nucleic acid recognition sequences relative to oneanother.
 33. A method for identifying a target nucleic acid molecule ina sample, which molecule is a member of a class of nucleic acidmolecules characterised by a specific variant region sequence, saidmethod comprising (i) contacting said sample with a nucleic acidmolecule according to any one of claims 1 to 29 for a time and underconditions sufficient to facilitate interaction of said nucleic acidmolecule with said target nucleic acid molecule; (ii) amplifying saidnucleic acid target; and (iii) optionally consecutively repeating saidamplification steps utilising the nucleic acid material amplified in thepreceding step together with a second generation nucleic acid moleculeaccording to claim 30 to 32; and (iv) detecting said amplified product.34. A method for detecting and/or monitoring a clonal population ofcells in a mammal, which clonal cells are characterised by a targetnucleic acid molecule which is a member of a class of nucleic acidmolecules characterised by a specific variant region sequence, saidmethod comprising: (i) contacting the nucleic acid material of abiological sample derived from a mammal with a nucleic acid moleculeaccording to any one of claims 1 to 29 for a time and under conditionssufficient to facilitate interaction of said nucleic acid molecule withsaid target nucleic acid molecule; (ii) amplifying said nucleic acidtarget; (iii) optionally consecutively repeating said amplificationsteps utilising the nucleic acid material amplified in the precedingstep together with a second generation nucleic acid molecule accordingto claim 30 to 32; and (iv) detecting said amplified product.
 35. Themethod according to claim 34 wherein said clonal population of cells isa neoplastic population of cells.
 36. The method according to claim 35wherein said neoplastic population of cells is a population ofneoplastic lymphoid cells.
 37. A method for the diagnosis of the onsetof or a predisposition to the onset of a disease condition or formonitoring or prognosing the progression of a disease condition in amammal, which condition is characterised by the presence or change inthe level of a target nucleic acid molecule, or clonal cell populationcharacterised by a target nucleic acid molecule, which molecule is amember of a class nucleic acid molecules characterised by a specificvariant region sequence, said method comprising: (i) contacting a samplederived from said mammal with a nucleic acid molecule according to anyone of claims 1 to 29, for a time and under conditions sufficient tofacilitate interaction of said nucleic acid molecule with said targetnucleic acid molecule; (ii) amplifying said nucleic acid target; (iii)optionally consecutively repeating said amplification steps utilisingthe nucleic acid material amplified in the preceding step together witha second generation nucleic acid molecule according to claim 30 to 32;and (iv) detecting said amplified product.
 38. The method according toclaim 37 wherein said disease condition is a neoplastic condition andsaid clonal population of cells is a neoplastic population of cells. 39.The method according to claim 38 wherein said neoplastic population ofcells is a population of lymphoid cells.
 40. The method according toclaim 37 wherein said condition is a microorganism infection and saidmicroorganism is a particular species or variant.
 41. The methodaccording to any one of claims 33 to 40 wherein said nucleic acidmolecule and second generation nucleic acid molecule areoligonucleotides.
 42. The method according to claim 41 wherein saidoligonucleotide is a primer.
 43. The method according to claim 42wherein said primer is a DNA primer.
 44. The method according to claim43 wherein the nucleic acid recognition region of said DNA primercomprises 3 nucleotides.
 45. The method according to claim 43 whereinthe nucleic acid recognition region of said DNA primer comprises 4nucleotides.
 46. The method according to any one of claims 33 to 39 or41 to 45 wherein said target nucleic acid molecule is a rearrangedgenomic T cell receptor gene.
 47. The method according to any one ofclaims 33 to 39 or 41 to 45 wherein said target nucleic acid molecule isa rearranged genomic immunoglobulin receptor gene.
 48. The methodaccording to claim 46 or 47 wherein the substantially conserved portionis the 3′ end of the V gene segment.
 49. A kit for facilitating theidentification of a target nucleic acid molecule, said kit comprisingcompartments adapted to contain any one or more of the nucleic acidmolecules according to claims 1 to 32, reagents useful for facilitatinginteraction of said nucleic acid molecule with the target nucleic acidmolecule and reagents useful for enabling said interaction to result inamplification of said nucleic acid target.
 50. The kit according toclaim 49 when used in the method of any one of claims 33 to 48.